Investigation of LAMP Technique in Diagnosis Type of Plasmodium Species in Anopheles Mosquitoes :A Fast and Practical Technique to Detect Malaria Pathogens in the Field

Background Malaria is one of the main parasitic diseases and a major health issue in some countries. This study aims to determine the rate and type of infections of Anopheles mosquitoes with malaria parasites using the molecular LAMP method in the Southeastern Iran. Methods In this study, 400 Anopheles mosquitoes were collected by the Zahedan Medical Insecticide Center in Nikshahr City, a high-risk area of malaria transmission in Sistan-Baluchestan Province. The mosquitoes were caught manually (by hand) in domestic (humans and animals), natural, and artificial outdoor places (Shelter pits). After DNA extraction, the LAMP method was used, which was compared with Multiplex Nested-PCR as a standard method. Results Out of 400 samples collected from Nikshahr City, 6 samples (1.5%) were infected with Plasmodium vivax. No Plasmodium falciparum or a mix (Plasmodium vivax and Plasmodium falciparum) was detected in this study. Conclusions The results of this study indicate that in places with transmission of both species, i.e. Plasmodium vivax and Plasmodium falciparum, detection of malaria parasites by the LAMP method could be very useful in spotting infections in the field. Thus, molecular epidemiological studies could be conducted annually to monitor malaria in endemic regions. The results of this research show that contamination with mosquito malaria vectors is increasing in Nikshahr City, and it seems that more studies will be required to eliminate malaria until 2026.


INTRODUCTION
Malaria is a critical public health issue in tropical and subtropical regions. In humans, four protozoan parasites, including Plasmodium vivax, P. falciparum, P. ovale, and P. malariae lead to the spread of this mosquito-borne infectious disease. The first antimalarial drugs is based on thorough knowledge of pathogens (1)(2)(3). Therefore, proper identification of pathogens is important before standard chemotherapy. According to a report released by the World Health Organization in 2016, despite various malaria control and antimalarial programs, 216 million cases of malaria were reported from 91 countries, which exceeded the number in 2016 by about 5 million cases. The annual mortality rate of malaria is around 445,000 individuals worldwide. Malaria deaths declined from 2015 to 2016 in Southeastern Asia but increased in Africa, Eastern Mediterranean, and the Pacific region (4,5) Iran is one of the known malaria endemic places in the Eastern Mediterranean region, with its endemic malaria areas being the provinces of Sistan-Baluchestan and Hormozgan, as well as Jiroft and Kahnuj Cities in Kerman Province (6) In Iran, the main goal of the malaria elimination program is to stop local transmission of the disease and to become a malaria-free region in 2026 (7). One of the essential strategies in the Global Malaria Control Program is the control of Anopheles mosquitoes, which is considered one of the most effective ways to eradicate and reduce malaria transmission around the world. Besides, awareness of the diversity of Anopheles species and infection with sporozoites in their salivary glands is one of the most decisive epidemiological steps in planning malaria control and prevention methods appropriate for local conditions (8)Various biochemical, molecular, and immunological methods(9-11), such as isoenzyme electrophoresis, polymerase chain reaction (PCR), and monoclonal antibodies have been used to detect Plasmodium in mosquitoes (12,13). One of the most common detection methods for malaria parasites in Anopheles mosquitoes is dissection of salivary glands in female Anopheles, captured by stereomicroscopy and light microscopy. However, this method is very cumbersome, time-consuming, and lowly sensitive (12,14). PCR methods are highly sensitive and specific, capable of detecting 1-10 parasites per microliter of blood, being five times more effective than microscopic detection by Giemsastained spreads. In addition to detecting parasite species, PCR methods could detect multiple infections.
Independent PCR detection of all pathogens with corresponding single primers needs more time, effort, and costs in the case of a mixed infection. Multiplex PCR-based approaches are more effective, in which more than one pair of primers are employed in a single response, thereby resulting in different sizes of DNA fragments being amplified simultaneously. In addition, they cut costs, save time, and are effective in detecting mixed infections. Thus, a multiplex nested-PCR method has been employed in the present study for simultaneous detection of P. vivax and P. falciparum (12). High sensitivity and specificity of this technique indicate its effectiveness in diagnosing asymptomatic malaria cases of a low parasitic burden. Besides, it is a perfect method for detecting malaria in low-transmission areas, which could be used in monitoring drug resistance as well. Furthermore, many samples could be tested simultaneously (12).
Loop-mediated isothermal amplification (LAMP) has made it possible to use a specific, rapid, sensitive, and simple technique for diagnosing a variety of diseases, including vector-borne ones (15). Bst DNA polymerase and a set of four specially designed primers, which identify six separate regions of the target DNA, are employed by LAMP. The target gene could be amplified and detected in an isothermal phase (15)(16)(17).Upon accumulation of about 10 9 copies of target DNA in less than one hour, auto-cycling strand displacement DNA synthesis is performed. A series of stem-loop DNA structures of different lengths are included in amplified products (18,19). Simple detection could be performed by visually inspecting turbidity of magnesium pyrophosphate, i.e. a byproduct of DNA synthesis, produced in proportion to the amount of amplified DNA (20,21). Using a loopamp real-time turbid meter, real-time detection could be performed as well (22). Due to the relatively favorable climate and geographical conditions in the southern and southeastern regions of Iran as well as migration to and from Afghanistan and Pakistan, malaria is a major public health issue in Iran . Sistan-Baluchestan Province has a long border with Pakistan, across which the largest number of malaria cases is observed (23). This study aims to examine the rate and type of Anopheles mosquito infections with sporozoites by Multiplex Nested-PCR and LAMP, which is performed in Nikshahr City of Sistan-Baluchestan Province. Anopheles mosquitoes were captured ( Figure 1). The Anopheles mosquitoes were caught manually (by hand) in domestic (humans and animals), natural, and artificial outdoor sites (Shelter pits), with suction tubes or aspirators utilized. All aspirating specimens were killed by a small chloroform-impregnated cotton pad placed in plastic cups. Just after the collection task, the samples were placed in 70% alcohol-containing tubes and transferred to the relevant health center. Using a high magnification entomology loop, the blood-fed Anopheles mosquitoes were separated and transferred into 1.5 ml tubes. Next, each sample was assigned a three-letter code and then stored in the tubes at -20 °C until DNA extraction.

Figure 1: Geographical locations where this study was carried out
Multiplex Nested-PCR Method: DNA extraction was performed by the column method through employing a DNA extraction kit (Produced by the DynaBio Blood/Tissue Genomic DNA Extraction Kit). Multiplex Nested-PCR was performed to determine species of Plasmodium via employing designed primers (Table 1). A pair of oligonucleotide primers (rPLU6 and rPLU5) was employed for the PCR-1 reaction in this study. In this reaction, primers, including rFAL.1, rFAL.2, rVIV.1, and rVIV.2 were used to simultaneously determine Plasmodium species (13,24). The first PCR product was diluted at a 1:100 ratio with distilled water as a template in the second PCR.
The amplified products were electrophoresed on 2% agarose gels in Tris-borate-EDTA for P. vivax and P. falciparum and then by the ultraviolet Trans illuminator stained with a red gel for visual detection.
The PCR steps included denaturation of the DNA molecule at 95 °C for 5 min and at 94 °C for 30 s, with 25 cycles for the first step and 30 cycles for the Nested-PCR, annealing at 58 °C for 1 min, extension at 72 °C for 60 s for the first step and 30 seconds for the Nested-PCR, final extension at 72 °C for 10 min for the first step and 5 min for the Nested-PCR, and finally, completion of the reaction and a reduction in the temperature to 20 °C (22,25,26). Table 1: Sequences of the primers used in the study for representation of the Plasmodium ssrRNA genes and multiplex/nested PCR protocol LAMP reaction: Table 2 provides information about the LAMP primer for amplifying gene coding for 18S rRNA specific Plasmodium genus (27,28). The LAMP reaction mixture (25  µL Table 3. The amount of reagents was computed based on the total number of the samples, which was provided as a master mix. Next, 24 µl of the mixture was poured separately into the new microtubes, and 1 µl of each sample's DNA was added next to each microtube. The reaction mixture was incubated in a water bath for 60 min at 60 °C and inactivated for 2 min at 80 °C. SYBR Green I (Invitrogen Carlsbad, CA, USA) was added to the reaction microtubes, and the resulting amplicons were spotted by visual observation. By triggering the reactions for 30, 45, 60, and 75 min at 60-64 °C in duplicate, optimal time and temperature conditions for the LAMP assay were defined. Besides, a LAMP reaction would be considered positive for Plasmodium spp if naked eyes detected an apparent rise in turbidity as opposed to the negative control. Two researchers assessed the results blindly.

DISCUSSION
Malaria is still a leading health issue in the southeastern regions of Iran, especially in Sistan-Baluchestan Province. In addition, it is effective in preventing economic and social growth in these deprived regions (29). In the present study, two molecular methods, including Multiplex Nested-PCR and LAMP, were used for the first time in Iran to detect malaria infections in vector mosquitoes. The mentioned two methods were highly sensitive, identified the genus and species of the parasites, and could detect mix infections. The current study was conducted in Nikshahr City, one of the high-risk areas of malaria transmission in Sistan-Baluchistan Province. In this study, a total of 400 blood-fed female Anopheles mosquitoes were captured. The results of this study showed that the dominant species of Anopheles mosquitoes in the studied area was A. culicifacies (83.33%).
Plasmodium vivax is the most prevalent malaria species in Iran, which is confirmed by contamination of 1.5% of the mosquitoes in Nikshahr City, being in line with findings of other studies (30). A similar study was conducted in Chabahar and Iranshahr by Oshaghi et al (2004), in which Plasmodium vivax was found to be the predominant species in the captured blood-fed female mosquitoes (31). Assmar et al (2005) conducted a study in endemic regions of Iran using PCR, and the results of which showed that Plasmodium vivax was the dominant parasite in the studied regions (32). Kalantari et al (2017) conducted a study in Fars Province, in which PCR identified a total of 512 female Anopheles mosquitoes. The results showed that Anopheles dthali (71.38%) was the dominant species in the mentioned region, yet no Plasmodium parasite infection was reported by mosquito PCR. However, in the present study, infections (1.66%) were reported in Anopheles mosquitoes. Sistan-Baluchestan Province is one of the endemic regions of malaria, with the difference between its endemic status and that of Fars province having caused the present study to report Plasmodium parasitic infections in A. stephensi (33).
In a similar study in India, 523Anopheles mosquitoes were studied to detect infection with Plasmodium. The results of this study showed that Anopheles annularis was the predominant species, yet no sporozoites were observed after salivary gland separation and microscopic examination. However, 7 Anopheles annularis mosquitoes (3.4%) were infected with Plasmodium falciparum by PCR (34). In another study in the Western India, Malaria infection in Anopheles was investigated by the Nested-PCR method, according to which, 2.8% of Anopheles subpictus were found to be contaminated with a mix of Plasmodium falciparum and vivax (35).
Many factors influence prevalence of Plasmodium species in different regions of the world. Climatic and ecological differences could be very effective in this regard. Determining the composition of Anopheles species and the amount of sporozoites in their salivary glands by reliable and sensitive methods is crucial for malaria prevention plans (8).
This study shows that the Multiplex Nested-PCR method could be effectively used for a large number of samples. This procedure does not require to dissect salivary glands, yet it can even examine dried and old specimens. Therefore, there is no need for equipping laboratories and dispatching personnel to malaria endemic regions because samples collected in these regions could be tested at central laboratories. On the other hand, the LAMP method employed for the first time in Iran to detect malaria in mosquitoes is simple, sensitive, and practical, which needs no expensive facilities and equipment. Besides, it could be used with a thermos of boiling water to easily detect contaminants in the field without wasting any time.
Afghanistan and Pakistan are recognized as the most dangerous countries for malaria transmission. Accordingly, since a large number of job seekers in Sistan-Baluchestan Province, especially in NikShahr City, are from these two countries, Iran remains on the list of malariaendemic countries (3,30,36). In a study in Konarak County, the reason for focal changes of malaria and its increasing incidence in 2008 was found to be presence of aliens (37). In another research, Raisi et al. reported that most non-indigenous malaria cases in Iran's Sistan-Baluchestan Province were emanated from bordering cities of Baluchestan State of Pakistan (38). In addition, Poudat et al. investigated factors affecting a malaria reduction in Bandar Abbas City and concluded that the natives of that city were always at the risk of malaria epidemics due to the presence of immigrants and movement of aliens (39). Therefore, the importance of migration as a major source of the crisis and a major challenge in controlling malaria should be considered.
Epidemiological investigations show that malaria in Iran, unlike that in Africa and other countries, is subject to climatic conditions and on the verge of eradication (25,26). This implies that this unstable state could contribute to the concern that increased rainfalls in high-risk regions could be associated with the higher prevalence of the disease (40). Climatic conditions, socioeconomic and cultural factors, quantity and quality of malaria control programs, common borders with Afghanistan and Pakistan, and issues related to Afghan and Pakistani immigrants are causative factors in the annual incidence of the disease in Sistan-Baluchestan Province (36). For the reasons mentioned above and due to the fact that Sistan-Baluchestan province is in the course of eliminating malaria, one of the most effective ways to monitor the abovementioned program is infection observation in Anopheles mosquitos. The results of this study, as against past studies, showed that malaria vector mosquito infection in Nikshahr City was increasing. Furthermore, it seems that elimination of malaria until 2026 requires further research.
The results show that in places with transmission of both species, i.e. Plasmodium vivax and Plasmodium falciparum, multiplex nested-PCR could be very effective in diagnosing malaria parasites for detection of mixed cases of the disease in both humans and vector mosquitoes. On the other hand, according to the results, the molecular LAMP method is very useful in detecting infections in the field due to its high sensitivity, short test time, and the need for inexpensive equipment. According to the Kappa's measure of agreement, both methods are fully consistent with each other (К=1). Thus, molecular epidemiological studies must be conducted annually to monitor this disease in endemic regions. According to the studies mentioned above and the present one, malaria vector mosquito infection is increasing in Nikshahr City. Given that Sistan-Baluchestan Province is in the course of eliminating malaria, failure to control this disease could lead to an epidemic.
Accordingly, continuous and substantial control measures are required to be implemented in all regions of the country. Furthermore, all border entry points need to be controlled, with illegal immigrants required to be prevented from entering the country.